Transcriptional regulation of flavonol biosynthesis in plants

Abstract Flavonols are a class of flavonoids that play a crucial role in regulating plant growth and promoting stress resistance. They are also important dietary components in horticultural crops due to their benefits for human health. In past decades, research on the transcriptional regulation of flavonol biosynthesis in plants has increased rapidly. This review summarizes recent progress in flavonol-specific transcriptional regulation in plants, encompassing characterization of different categories of transcription factors (TFs) and microRNAs as well as elucidation of different transcriptional mechanisms, including direct and cascade transcriptional regulation. Direct transcriptional regulation involves TFs, such as MYB, AP2/ERF, and WRKY, which can directly target the key flavonol synthase gene or other early genes in flavonoid biosynthesis. In addition, different regulation modules in cascade transcriptional regulation involve microRNAs targeting TFs, regulation between activators, interaction between activators and repressors, and degradation of activators or repressors induced by UV-B light or plant hormones. Such sophisticated regulation of the flavonol biosynthetic pathway in response to UV-B radiation or hormones may allow plants to fine-tune flavonol homeostasis, thereby balancing plant growth and stress responses in a timely manner. Based on orchestrated regulation, molecular design strategies will be applied to breed horticultural crops with excellent health-promoting effects and high resistance.


Introduction
Flavonols belong to one class of f lavonoids with the C 6 -C 3 -C 6 basic structure and are characterized by the carbon-carbon bond (C2 and C3 positions), a hydroxyl group (C3 position), and a carbon group (C4 position) in the heterocyclic C-ring (Fig. 1a).Due to the varying substitutions of hydroxylation and methoxylation on the A and B rings, f lavonol aglycones can be classified into more than 10 different types [1].Among them, kaempferol, quercetin, and myricetin are the most prevalent f lavonol aglycones in the plant kingdom [1,2] (Fig. 1a).Flavonols are commonly found in glycosylated forms and are ubiquitously distributed in various plant tissues [1,2].They play a vital role in plant development and stress resistance, including regulating auxin transport, affecting root and pollen development, inf luencing pollinator preference and reproductive isolation, UV-B protection, and enhancing disease resistance [3][4][5][6][7][8].
Until now, more than 26 000 articles can be retrieved from the PubMed database (https://pubmed.ncbi.nlm.nih.gov/) by using the keyword 'quercetin', one class of f lavonol aglycone.In the past few decades, significant progress has been made in understanding the transcription factors (TFs) and microRNAs (miRNAs) that regulate f lavonol biosynthesis in plants.However, to date there has been no systematic review of the transcriptional regulation of f lavonol biosynthesis in plants.
This review focuses on recent advances in knowledge and understanding of the transcriptional regulation of f lavonol biosynthesis in plants.Progress in the characterization of TFs and  miRNAs regulating f lavonol biosynthesis will be summarized, with particular emphasis on direct transcriptional regulation and cascade transcriptional regulation of f lavonol biosynthesis.
Direct transcriptional regulation involves TFs, such as MYB, AP2/ERF, and WRKY, which directly target the key FLS gene and other early genes in the f lavonoid biosynthesis pathway.In addition, different regulation modules in cascade transcriptional regulation will be summarized and discussed; these consist of miRNAs targeting TFs, regulation between activators, interaction between activators and repressors, and degradation of activators or repressors induced by environmental signals such as UV-B or plant hormones.This review may provide valuable insights for the production of horticultural crops with a high content of f lavonols that are beneficial for human health.
Flavonol aglycones finally undergo modifications by uridine diphosphate-glycosyltransferases (UGTs), O-methyltransferases (OMTs), and acyltransferases (ATs), leading to the formation of various and stable f lavonol derivatives [34][35][36].In addition, dihydrof lavonols also can be acted on by dihydrof lavonol 4reductase (DFR) to produce leucoanthocyanidins and to direct f lavonoid metabolism to the synthesis of anthocyanins and proanthocyanidins.The competition between FLS and DFR regulates metabolic f lux to different branches of the f lavonoid biosynthetic pathway [37].

R2R3-MYB transcription factors
The MYB (V-myb avian myeloblastosis viral oncogene homolog) family is widely present in all eukaryotes and represents one of the largest TF families in plants.The N-terminus of MYB family proteins contains a highly conserved DNA-binding domain which typically consists of one to four imperfect repeats [38].Each repeat of ∼50-53 amino acids forms a helix-turn-helix structure, allowing them to bind to the major groove of the DNA double helix [39].MYB proteins are classified into four types: MYB-related (R1/2-MYB, R3-MYB), R2R3-MYB, 3R-MYB (R1R2R3-MYB), and 4R-MYB [38,40].R2R3-MYB TFs are the largest class in plants and are composed of two DNA-binding repeats.Based on phylogenetic relationships and the presence of conserved motifs in the Cterminal region, R2R3-MYB proteins are classified into different subgroups, including subgroup 4 (SG4), SG7, SG19, etc. [38,40,41].In past decades there has been an increasing number of studies focusing on R2R3-MYB TFs involved in regulating f lavonol biosynthesis (Fig. 1b and Table 2).SG7 R2R3-MYBs.The SG7 R2R3-MYBs are f lavonol-specific regulators with the characteristic SG7 (GRTxRSxMK) motif and have been characterized in numerous plants.In Arabidopsis thaliana, AtMYB12, along with its homologs AtMYB11 and AtMYB111, belongs to SG7 of the R2R3-MYB family.These proteins controlled f lavonol biosynthesis by independently activating expression of AtCHS, AtCHI, AtF3H, and AtFLS1 [42,43].MdMYB22 from apple and CsMYB12 from tea are the homologs of AtMYB12 and have been shown to act as activators via binding to the FLS promoter and activating its transcription in vivo, as confirmed through yeast one-hybrid and luciferase assays [44,45].In Freesia hybrida, chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) and β-glucuronidase assays indicated that FhMYB1/2/3/4 bind to MYBCORE and ACrich elements in promoters of FhCHI2 and FhFLS1 to activate their transcription [46].Furthermore, MrMYB12 from Chinese bayberry (Morella rubra) was found to bind to the MYBCORE element in the MrFLS2 promoter and activate its expression, as demonstrated by EMSA and luciferase assays [47].These results demonstrate that SG7 R2R3-MYBs can directly target the key gene FLS and other early genes in the f lavonoid biosynthetic pathway.
In addition, secondary metabolite profiling analyzed by LC-MS showed a selective reduction of glycosylated f lavonol derivatives in single, double, or triple mutants of Arabidopsis AtMYB11, AtMYB12, and AtMYB111 [43].Meanwhile, the accumulation of other phenolic compounds in the mutant seedlings remained significantly unchanged [43].In tomato, according to an LC-MS analysis, levels of 13 glycosylated f lavonol derivatives, quercetin, naringenin, and naringenin chalcone were reduced in pf mutants with truncation of SlMYB12 [48].In Petunia axillaris, a myb-f l CRISPR mutant strongly reduced f lavonol levels and expression of FLS and HT1 (F3 H) [49].In addition, overexpression of one SG7 R2R3-MYB gene, such as AtMYB12 [37], Gentiana trif lora GtMYBP3/4 [50], Epimedium sagittatum EsMYBF1 [51], peach (Prunus persica) PpMYB15/PpMYBF1 [52], or Chinese bayberry MrMYB12 [53], resulted in f lavonol accumulation in tobacco (Nicotiana tabacum) flowers.This was caused by upregulated expression of NtFLS and other early genes in the f lavonoid biosynthesis pathway.Transgenic tobacco f lowers changed from red to pale or pure white and showed a reduction in anthocyanin content, which was not consistent with the unaffected expression of NtDFR and other anthocyanin biosynthetic genes [37,50,52,53].Such apparent changes in f lower color suggest that FLS may effectively compete with DFR to redirect the f lux towards f lavonol biosynthesis and away from anthocyanin biosynthesis.These findings indicate that SG7 R2R3-MYB TFs may be f lavonol-specific activators in plants.
High expression of SG7 R2R3-MYB genes may be one of reasons for high accumulation of f lavonols in horticultural crops such as tea, apple, and Chinese bayberry (Table 1).In tea, high expression of CsMYB12 in the first leaf or UV-B-irradiated leaf resulted in a high accumulation of quercetin glycosides and kaempferol glycosides [45].In apple, expression of MdMYB22 was positively correlated with the f lavonol content in fruit of F 1 hybrid populations of a cross between Malus sieversii f. niedzwetzkyana and M. domestica [44].In Chinese bayberry, the transcript level of MrMYB12 was induced by UV-B irradiation and was correlated with high accumulation of quercetin derivatives [47].SG19 R2R3-MYBs.In addition to SG7 R2R3-MYBs, SG19 R2R3-MYB TFs were also found to be involved in the positive regulation of f lavonol biosynthesis by activating transcription of the FLS gene.In F. hybrida, an SG19 R2R3-MYB protein, FhMYB21L2, could directly target and regulate one FLS member, FhFLS2, thus participating in f lavonol accumulation in later developmental stages of the f lower [46].On the other hand, four SG7 R2R3-MYB members, namely FhMYBF1/2/3/4, were found to regulate expression of FhFLS1 and f lavonol biosynthesis in early developmental stages of the f lower [46].In Arabidopsis, SG19 R2R3-MYB members, including AtMYB21/24/57, have also been shown to be involved in regulating f lavonol biosynthesis by controlling expression of AtFLS1 [46,71].An SG19 R2R3-MYB TF, MdMYB8, was also identified in Malus crab apple and was responsible for quercetin 7-O-glucoside accumulation by regulating expression of MdCHS and MdFLS [72].
Other R2R3-MYBs.SG4 R2R3-MYB members contain an EAR motif (LNL[D/E]L) and have been identified as negative regulators of f lavonols and other phenolic compounds.In Arabidopsis, the atmyb4 atmyb7 mutant showed an increase in the accumulation of f lavonols and anthocyanins, caused by induced expression of general phenylpropanoid pathway genes, such as C4H, 4CL1, etc. [73].In Tartary buckwheat, SG4 R2R3-MYB members FtMYB14/15/16 could directly target FtPAL gene and inhibit activity of its promoter, thereby negatively regulating rutin biosynthesis [74,75].FtMYB11 and FtMYB13 are non-typical R2R3-MYB repressors that do not belong to SG4 R2R3-MYB while their function is similar to that of FtMYB14/15/16 [74,76].These SG4 R2R3-MYBs target and negatively regulate the general phenylpropanoid pathway genes.
In addition, AtMYB13 is an SG2 R2R3-MYB and could activate promoters of AtCHS, AtCHI, and AtFLS1, thereby enhancing f lavonol accumulation in Arabidopsis seedlings [7].In Chinese bayberry, two SG44 R2R3-MYB proteins, MrMYB5 and MrMYB5L, could activate transcription of MrF3 5 H and MrFLS1 by binding to their promoters, based on EMSA and luciferase assays [47].MrMYB5 or MrMYB5L also interacted with MrbHLH2 to synergistically regulate expression of MrF3 5 H and MrFLS1, and these MYB-bHLH protein complexes play a crucial role in regulating myricetin biosynthesis [47].This specific transcriptional mechanism of f lavonol biosynthesis might be the reason for the high accumulation of myricetin derivatives in fruit and leaf of Chinese bayberry (Table 1).

WRKY transcription factors
The WRKY proteins have been shown to be regulators of f lavonol biosynthesis, including activators and repressors (Fig. 1b and Table 2).In apple, overexpression of MdWRKY11 promoted f lavonoid accumulation and upregulated expression of MdF3H, MdFLS, MdDFR, MdANS, and MdUFGT in apple calli [77].In tobacco, chromatin immunoprecipitation assays and overexpression experiments demonstrated that NtWRKY11b could target and activate promoters of NtMYB12, NtFLS, NtGT5, and NtUFGT, thereby inducing f lavonol accumulation [78].Overexpression of VqWRKY31 from Vitis quinquangularis in grape increased accumulation of f lavonoids and stilbenes and promoted expression of VvCHS, VvCHI, VvDFR, VvFLS, and VvSTS (stilbene synthase), which enhanced powdery mildew resistance [79].Moreover, VvWRKY70 was identified as a transcriptional repressor of f lavonol biosynthesis in grape by inhibiting the transcriptional activity of VvFLS4 and VvCHS2/3 [80].Overexpression of VvWRKY70 caused a reduction of f lavonol contents in transgenic grape calli [80].These WRKY TFs can directly regulate expression of FLS genes to participate in regulation of f lavonol biosynthesis.
In addition, there were other WRKY activators involved in regulation of f lavonol accumulation by activating early genes in f lavonoid biosynthesis (Fig. 1b and Table 2).For example, RNAi and overexpression experiments demonstrated that Arabidopsis AtWRKY23 functioned as a positive regulator of f lavonol biosynthesis by activating AtF3 H expression and was also required for proper root growth and development [5].In cotton (Gossypium hirsutum), ChIP-qPCR, yeast two-hybrid, biomolecular f luorescence complementation, and firef ly luciferase complementation imaging assays revealed that GhWRKY41could form a homodimer with itself to directly activate GhWRKY41 itself and expression of GhC4H and Gh4CL, which promoted accumulation of f lavonoids and lignin to improve cotton resistance to Verticillium dahliae [81].Overexpression of VqWRKY56 from Vitis quinquangularis increased f lavonoid content by directly targeting VvCHS3 and other f lavonoid biosynthetic genes in the transgenic grape leaf, which reduced susceptibility to powdery mildew [82].

bZIP transcription factors
A well-studied example is Arabidopsis ELONGATED HYPOCOTYL 5 (HY5), a bZIP TF that activates expression of AtCHS, AtFLS, and other genes to regulate f lavonoid accumulation during photomorphogenesis in seedlings [83].In recent years, several newly discovered bZIP activators have also been found to be involved in positive regulation of f lavonol biosynthesis (Fig. 1b and Table 2).In Populus tremula × P. alba, overexpression and suppression experiments indicated that PtabZIP1L could positively regulate f lavonoid accumulation by affecting expression of PtaFLS2/4, which mediated lateral root development and drought resistance [84].In grape, CRISPR/Cas9-mediated mutagenesis of VvbZIP36 promoted anthocyanin accumulation but inhibited f lavonol biosynthesis in the leaf, which was associated with upregulation of anthocyanin biosynthetic genes and downregulation of VvFLS2/4 and two VvFLR (f lavonol-3-Orhamnosyltransferase) genes, respectively [85].Overexpression of grape VvibZIP22 in tobacco promoted accumulation of f lavonols and anthocyanins and induced expression of NtPAL, NtCHS, NtDFR, and NtANS [86].In rice, OsbZIP48 was identified as a positive regulator of f lavonoid biosynthesis through a metabolitebased genome-wide association study [87].Yeast one-hybrid and luciferase assays further demonstrated that OsbZIP48 could directly bind to promoters of Os4CL5 and OsCHS and activate their transcription [87].Interestingly, pear PpbZIP44 could positively regulate expression of PpF3H and PpADT, which encodes an enzyme, arogenate dehydratase, of primary metabolism as a key determinant of carbon f low into the phenylpropanoid pathway [88].Therefore, transient overexpression of PpbZIP44 in pear fruit promoted accumulation of phenylalanine and f lavonoids [88].

AP2/ERF transcription factors
In recent years, several AP2/ERF TFs have been reported to be involved in positive or negative regulation of f lavonol biosynthesis (Fig. 1b and Table 2), but it is unknown whether these ERFs are regulated by ethylene signals.In tomato, overexpression of SlERF.G3like activated expression of SlFLS and other early genes in the f lavonoid biosynthesis pathway, such as SlCHS1/2, SlCHI, SlF3H, and SlF3 H, which resulted in induction of f lavonol content in fruit [89].SlERF.G3-like appeared to act independently of the f lavonolspecific activator SlMYB12 [89].Overexpressing MdAP2-34 in apple callus induced f lavonol accumulation by targeting and activating the MdF3 H promoter [90].Overexpression of citrus CsERF003 in tomato led to accumulation of f lavonol glycosides and naringenin chalcone by activating expression of SlPAL, SlC4H, Sl4CL, SlCHS, SlCHI, SlF3 H, and SlFLS [91].In addition, an ERF transcription repressor, FtERF-EAR3, was identified to inhibit FtF3H expression and f lavonol biosynthesis by binding to the GCC-box in the FtF3H promoter in Tartary buckwheat [92].

Other transcription factors
Members of other TF families have also been reported to be involved in regulation of f lavonol biosynthesis and can be divided into two classes (Fig. 1b and Table 2).First, TFs such as MdSCL8, AaYABBY5, and PtHSF5a directly target FLS and regulate its expression.In apple, 5-aminolevulinic acid (ALA) inhibited expression of MdSCL8, which alleviated its transcriptional repression of MdFLS1 and promoted f lavonol accumulation [93].In Artemisia annua, overexpression of AaYABBY5 upregulated expression of AaPAL, AaCHS, AaCHI, AaFLS, AaFSII, AaLDOX, and AaUFGT, resulting in a significant increase in total f lavonoid content [94].In Populus tomentosa, overexpression of PtHSFA5a upregulated expression of PtCHS1, PtF3 H2, and PtFLS1/2, leading to a significant increase in f lavonol content in the transgenic poplar [95].EMSA, ChIP-qPCR, and luciferase assays demonstrated that PtHSFA5a can directly bind to the promoters of PtCHS1 and PtFLS1 to enhance their transcription [95].Second, TFs act as regulators by targeting early genes in f lavonoid biosynthesis.In Arabidopsis, REPLUMLESS (RPL) TF was necessary for bacterial resistance and could repress f lavonol accumulation by inhibiting expression of the CHI gene, which regulated auxin transport to promote plant growth [96].In tobacco, overexpression and suppression experiments showed that an HD-ZIP IV TF, NtHDG2, could regulate f lavonol biosynthesis by targeting and activating promoters of NtF3 H and NtF3GT [97].In sweet potato (Ipomoea batatas), EMSA and ChIP-qPCR indicated that IbBBX29 could bind to specific T/G-boxes in the promoters of IbCHS1, IbCHI1, and IbF3 H to activate their expression [98].Overexpression of IbBBX29 increased contents of f lavonols and other f lavonoids by upregulating expression of f lavonoid biosynthetic genes in storage roots of sweet potato [98].

Cascade transcriptional regulation of flavonol biosynthesis
Post-transcriptional regulation miRNAs are a class of non-coding RNAs with lengths 20-24 nt and regulate post-transcriptional processes by recognizing target genes through base complementarity, leading to mRNA cleavage or translational inhibition [99].With the application and deep exploration of genomics, several miRNAs involved in f lavonol biosynthesis have been identified in plants (Fig. 1b and Table 2).The miR858-MYB regulatory modules have been reported to regulate f lavonol biosynthesis in plants.In Arabidopsis, transgenic experiments indicated that AtmiR858a directly targeted and cleaved SG7 R2R3-MYB genes, including AtMYB11/12/111, resulting in the negative regulation of f lavonol biosynthesis [100].Subsequent research revealed that primary AtMIR858a encoded a small peptide, miPEP858a, which was involved in transcriptional regulation of AtmiR858a [101].This peptide could negatively regulate f lavonol biosynthesis by inhibiting expression of AtMYB12 through AtmiR858a.In potato, overexpression of StmiR858 inhibited expression of StMYB12A/C genes, leading to a reduction in f lavonol accumulation [15].As research goes on, members of other miRNA families and their different target genes are continually discovered.In cotton, overexpression of GhSPL10, a target of GhmiR157a, resulted in a significant increase in f lavonol accumulation, which promoted initial cellular dedifferentiation and callus proliferation [102].In Arabidopsis, overexpression of apple MdmiR172 targeting MdAP2_1a reduced levels of anthocyanins and f lavonols as well as expression of AtFLS1 and other f lavonoid biosynthetic genes in plantlets, which may be caused by regulation of the MdAP2_1a-MdMYB10 module [103].Current research on miRNAs regulating f lavonol biosynthesis is quite limited, warranting further investigation.

UV-B regulation
Solar ultraviolet (UV) light consists of UV-A (320-400 nm) and a portion of UV-B (280-320 nm).Elevated UV-B radiation leads to the generation of numerous free radicals within plants, which induces damage to DNA, RNA, and proteins.Flavonols serve as efficient scavengers of free radicals and UV-B absorbers [4].Preharvest UV-B radiation was found to increase f lavonol accumulation in apple and grape fruits [104,105].In many studies UV-B radiation as a method of postharvest treatment was applied to improve f lavonol content in vegetables and fruits, such as apple [106], asparagus [107], broccoli [108], Chinese bayberry [47], cucumber [109], kale (Brassica oleracea var.sabellica) [110], mango [111], onion [112], peach [35], and tomato [113].Recently, significant progress has been made in the regulatory mechanisms of plant f lavonol biosynthesis in response to UV-B signal in UVR8-dependent and UVR8-independent ways, including R2R3-MYB and other non-MYB TFs (Fig. 2 and Table 2).UVR8-dependent UV-B signaling pathway.UV-B signal is perceived by the photoreceptor UV RESISTANCE LOCUS (UVR8), which is a plant-specific and highly conserved protein [114,115].UVR8 was inactive in its dimeric form in the absence of UV-B, and CONSTI-TUTIVELY PHOTOMORPHOGENIC 1 (COP1) induced ubiquitination and degradation of ELONGATED HYPOCOTYL 5 (HY5) by the 26S proteasome, thus repressing expression of downstream target genes [114,116] (Fig. 2).After UV-B perception, dimeric UVR8 underwent monomerization to form monomeric UVR8, which interacted with COP1 to form a complex that repressed COP1 activity [114,116].The central TF HY5 was ultimately stabilized and activated transcription of UV-B-responsive genes [116,117].
In Arabidopsis, HY5 could directly target and activate expression of AtMYB12 and AtMYB111 under UV-B radiation, leading to f lavonol accumulation in the seedlings [4,118] (Fig. 2).This HY5-SG7 R2R3-MYB regulatory module has also been identified in horticultural crops such as grape [119], apple [104], and tea [45].MdHY5 and MdMYB22 (a homolog of AtMYB12) from apple could also synergistically regulate transcription of MdCHS and MdFLS, leading to the induction of f lavonol accumulation under UV-B radiation [104].These results indicated that HY5 regulates f lavonol biosynthesis in two steps.First, it directly binds to promoters of SG7 R2R3-MYBs and induces their expression.Secondly, it can interact with SG7 R2R3-MYBs to synergistically regulate expression of FLS and other early genes in f lavonoid biosynthesis.In addition to the HY5-SG7 R2R3-MYB module, monomeric AtUVR8 in Arabidopsis could also directly interact with AtMYB13 to form a complex that enhanced the affinity of AtMYB13 for promoters of AtCHS, AtCHI, and AtFLS1 [7] (Fig. 2).This UVR8-MYB13 module further promoted f lavonol accumulation and plant resistance to UV-B stress [7].UVR8-independent UV-B signaling pathway.Apart from the UVR8dependent UV-B signaling pathway, the UVR8-independent stress response has been recently identified in plants (Fig. 2 and Table 2).Under white light conditions without UV-B radiation, the brassinosteroid signal led to activation of BRI1-EMS-SUPPRESSOR 1 (BES1) (a master TF in brassinosteroid signal transduction) in Arabidopsis [120].This activation of AtBES1 resulted in downregulation of AtMYB11, AtMYB12, and AtMYB111 expression, consequently leading to a decrease in f lavonol accumulation [120].However, when Arabidopsis plants were exposed to UV-B radiation, the UVR8-COP1-HY5 module was activated to initiate UV-B photomorphogenesis, including activation of SG7 R2R3-MYB expression [120].In addition, UV-B stress could also inhibit expression of AtBES1 in a UVR8-independent manner, which removed inhibition of SG7 R2R3-MYB expression and promoted f lavonol accumulation [120].The UV-B stress-induced inhibition of AtBES1 expression reallocated more energy towards f lavonol biosynthesis, which promptly shifted plants from brassinosteroid-promoted growth to UV-B stress response and ensured normal plant growth under adverse conditions.

Phytohormonal regulation
Jasmonates.Jasmonates (JAs) are vital plant hormones and trigger a cascade of stress-related gene expressions in response to biotic and abiotic stress through the signaling module of the SCF COI1jasmonate-ZIM domain (JAZ).Induction of f lavonol accumulation by JAs has been observed in plants such as blackberry (Rubus sp.) [121], G. biloba [122], and Tartary buckwheat [76].In Arabidopsis, JAZ proteins interfered with MYB-bHLH complexes, which were composed of IIIe-bHLH TFs (MYC2, MYC3, MYC4, and MYC5) and SG19 R2R3-MYB TFs (MYB21 and MYB24) [123].JA signals were perceived by CORONATINE-INSENSITIVE PROTEIN 1 (COI1), which recruited proteins to the Skp1/Cullin/F-box (SCF COI1 ) complex for ubiquitination and subsequent degradation by the 26S proteasome pathway [124,125].Thus, the degradation of JAZ proteins led to release of the MYB-bHLH complexes, which regulated expression of downstream genes involved in stamen development [123,126] (Fig. 3).A further study demonstrated that AtMYB21 and AtMYB24 activated transcription of AtFLS1 to induce accumulation of pollen-specific f lavonols, which enhanced reactive oxygen species (ROS) scavenging capacity and contributed to male fertility [71].
Auxin.Auxin is an important plant hormone for plant growth and development through the SCF TIR1 -IAA-ARF module [128].Auxin has also been reported to positively regulate f lavonol biosynthesis [129,130].In Arabidopsis, Lewis et al. [130] found that auxin could upregulate expression of AtMYB12, AtCHS, AtCHI, AtF3 H, and AtFLS through the Transport Inhibitor Response1 (TIR1) signaling pathway [130].They speculated that auxin response factors (ARFs) play a crucial role in this process [130] (Fig. 3).Recently, AtARF2 was identified as a positive regulator of f lavonol biosynthesis through directly activating transcription of the AtMYB12 and AtFLS genes [131].Another study indicated that auxin-induced f lavonol accumulation also depended on the ARF pathway [5] (Fig. 3 and Table 2).In Arabidopsis, auxin may mediate degradation of SOLITARY ROOT/INDOLE-3-ACETIC ACID14 (SLR/IAA14) by the SCF TIR1 complex, which led to release of AtARF7/19 and subsequent induction of AtWRYY23 expression, thereby activating AtF3 H expression to induce f lavonol biosynthesis in the roots [5,132].
Gibberellic acid.Gibberellic acid (GA) is an important plant hormone for plant growth and participates in negative regulation of f lavonol biosynthesis through the GID1-SCF SLY1/GID2 -DELLA sig-naling module [133][134][135].In plants, GA signals promoted the interaction between GID1 (GA-INSENSITIVE DWARF1) and DELLA proteins, enhancing the binding affinity of the GID1-DELLA complex with the SCF SLY1/GID2 complex.This interaction led to the degradation of DELLA proteins by the 26S proteasome pathway [133,134] (Fig. 3).Recently, a study in Arabidopsis revealed that GA negatively regulated f lavonol biosynthesis through the DELLA-SG7 R2R3-MYB module [135] (Fig. 3 and Table 2).In the absence of GA, DELLA protein accumulated and physically interacted with SG7 R2R3-MYBs, which enhanced the transcriptional activation activity of AtMYB12 and AtMYB111 on promoters of AtFLS1 and AtF3H [135].This promoted f lavonol biosynthesis in Arabidopsis roots and inhibited auxin transport and root growth.Conversely, GA signaling promoted degradation of the DELLA protein by the 26S proteasome pathway.Subsequently, this reduced the transcriptional activation activity of SG7 R2R3-MYB proteins and f lavonol content in Arabidopsis roots, which led to an increase of auxin accumulation in root tip cells and promotion of root growth [135].
Abscisic acid.Abscisic acid (ABA) as a plant hormone plays an important role in plant growth and fruit ripening [136].ABA can also modulate the stomatal aperture by directly promoting production of ROS in plant guard cells [137].Flavonols, as important ROS scavengers, are involved in the regulation of ABAinduced stomatal closure in plants [138,139].In tobacco, ABA treatment could inhibit expression of NtMYB184 (a f lavonolspecific activator), which reduced production of f lavonols and thus increased ROS levels to regulate stomatal closure [65].ALA, known as a new natural plant growth regulator, can reverse ABA-induced stomatal closure [140].In apple, ALA treatment enhanced protein abundance and phosphorylation of protein phosphatase 2AC (MdPP2AC), which promoted the interactions of different PP2A subunits and increased holoenzyme activity [140].Phosphorylated PP2A interacted with and dephosphorylated MdSnRK2.6 (sucrose non-fermenting 1-related protein kinase 2.6), which induced f lavonol accumulation and thus reduced ROS levels in the guard cells to open stomata [140].In addition, ALA treatment could inhibit expression of MdSCL8 (a f lavonol repressor), which may promote expression of MdFLS1 and f lavonol accumulation to participate in regulation of stomata opening [93].
Other regulation modules.Several members of other TF families were identified as repressors by negatively regulating transcriptional activity of f lavonol-related activators (Table 3).In Arabidopsis, AtMYB4 could repress activation of the AtCHS and AtFLS promoters by AtMYB12 and AtMYB111 [141].In tomato, loss-of-function and luciferase assays indicated that SlSPL-CNR functioned as a negative regulator of f lavonol biosynthesis by repressing SlMYB12 transcription activity [142].In P. tomentosa, yeast two-hybrid, pull-down, co-immunoprecipitation, and luciferase assays demonstrated that PtIAA17.1 could interact with PtHSFA5a to suppress PtHSFA5a-mediated activation of PtCHS1 and PtFLS1 [75].Salt stress enhanced the stability of PtIAA17.1, resulting in the promotion of its interaction with PtHSFA5a and the repression of f lavonol biosynthesis [95].In addition, a receptor-like kinase (OsRLCK160) could regulate f lavonoid accumulation in rice by interacting with and phosphorylating OsbZIP48 [87].

Conclusions and perspective
Flavonols are an important branch of the f lavonoids with excellent bioactive activities, and they are abundant in horticultural crops.Due to the broad function of f lavonols in plants, sophisticated regulation networks involving different types of TFs (activators and repressors) and miRNAs have evolved, illustrating fine-tuned f lavonol homeostasis under specific environmental conditions or hormonal signals.Significant progress has been achieved in unraveling the transcriptional regulation of f lavonol biosynthesis in Arabidopsis and some horticultural crops, such as tea, apple, and Chinese bayberry.However, more f lavonol-rich horticultural crops deserve in-depth investigations to discover novel TFs and elucidate the specific regulatory mechanisms of f lavonol biosynthesis.Besides transcriptional regulation, posttranscriptional regulation, post-translational modifications, and epigenetic regulation of f lavonol biosynthesis in plants are also worthy of further exploration.
In addition, f lavonol derivatives are associated with the astringency of horticultural crops.Increasing the f lavonol content of fruits and vegetables by genetic selection, genetic engineering, or physical treatment may be of interest for human health, but it might negatively affect the taste quality.Structural modification of secondary metabolites caused by decorations such as hydroxylation and glycosylation can alter the taste of the compounds.So far, there is limited research reporting the improvement of the f lavor quality of f lavonols by structural modification, which deserves future study.This will provide valuable insights for utilizing molecular design breeding and synthetic biology to enhance f lavonol accumulation in horticultural crops with significant bioactivities and unaffected f lavor quality, thereby facilitating the development of the horticultural industry.

Figure 2 .
Figure 2. Cascade transcriptional regulation of f lavonol biosynthesis in plants under UV-B radiation.The yellow box with bold letters means promotion of f lavonol biosynthesis, whereas the blue box with non-bold letters represents repression of f lavonol biosynthesis.Line thickness indicates the activity of transcriptional activation or repression.Light orange and blue-gray ovals represent activators and repressors, respectively.

Figure 3 .
Figure 3. Cascade transcriptional regulation of f lavonol biosynthesis in plants in response to different hormonal signals.Yellow boxes with bold letters mean promotion of f lavonol biosynthesis, whereas blue boxes with non-bold letters represent repression of f lavonol biosynthesis.Line thickness indicates the activity of transcriptional activation or repression.Light yellow and light gray backgrounds indicate activation and repression of f lavonol biosynthesis, respectively.Light orange and blue-gray ovals represent activators and repressors, respectively.JA, jasmonates; GA, gibberellic acid; Ub, ubiquitin.

Table 1 .
Flavonol content in the edible portion of staple crops, medicinal plants, and horticultural plants.

Species Content (mg/100 g FW) Reference Species Content (mg/100 g FW) Reference
Flavonols in plants are usually present in glycosylated forms.Therefore, the flavonol content was defined as the total content of flavonol glycosides.* Dry weight basis; FW, fresh weight; n.d., not detected.

Table 2 .
Different types of transcription factors or regulation modules involved in regulating f lavonol biosynthesis in plants.

Table 3 .
Different regulation modules in cascade transcriptional regulation of f lavonol biosynthesis in plants.